1. Field of the Invention
The present invention relates to a rapid method for the detection of ischemic states and to a kit for use in such a method. More particularly, the invention relates to the measurement of a bound specific transition element to human serum to determine the presence or absence of ischemia.
2. Discussion of the Background
Ischemia is the leading cause of illness and disability in the world. Ischemia is a deficiency of oxygen in a part of the body causing metabolic changes, usually temporary, which can be due to a constriction or an obstruction in the blood vessel supplying that part. The two most common forms of ischemia are cardiovascular and cerebrovascular. Cardiovascular ischemia, in which the body""s capacity to provide oxygen to the heart is diminished, is the leading cause of illness and death in the United States. Cerebral ischemia is a precursor to cerebrovascular accident (stroke) which is the third leading cause of death in the United States.
The continuum of ischemic disease includes five conditions: (1) elevated blood levels of cholesterol and other blood lipids; (2) subsequent narrowing of the arteries; (3) reduced blood flow to a body organ (as a result of arterial narrowing); (4) cellular damage to an organ caused by a lack of oxygen; (5) death of organ tissue caused by sustained oxygen deprivation. Stages three through five are collectively referred to as xe2x80x9cischemic disease,xe2x80x9d while stages one and two are considered its precursors.
Together, cardiovascular and cerebrovascular disease accounted for 954,720 deaths in the U.S. in 1994. Furthermore, more than 20% of the population has some form of cardiovascular disease. In 1998, as many as 1.5 million Americans will have a new or recurrent heart attack, and about 33% of them will die. Additionally, as many as 3 to 4 million Americans suffer from what is referred to as xe2x80x9csilent ischemia.xe2x80x9d This is a condition where no clinical symptoms of ischemic heart disease are present.
There is currently a pressing need for the development and utilization of blood tests able to detect injury to the heart muscle and coronary arteries. Successful treatment of cardiac events depends largely on detecting and reacting to the presence of cardiac ischemia in time to minimize damage. Cardiac enzymes, specifically the creatine kinase isoenzyme (CK-MB), and cardiac markers, specifically the Troponin I and T biochemical markers, are utilized for diagnosing heart muscle injury. However, these enzymes and markers are incapable of detecting the existence of an ischemic state in a patient prior to myocardial infarction and resulting cell necrosis (death of cell). Additionally, these enzymes and markers do not show a measurable increase until several hours after an ischemic event. For instance, CK-MB, the earlier evident of the two, does not shows a measurable increase above normal in a person""s blood test until about four to six hours after the beginning of a heart attack and does not reach peak blood level until about 18 hours after such an event. Thus, the primary shortcoming of using cardiac markers for diagnosis of ischemic states is that these markers are only detectable after heart tissue has been irreversibly damaged.
There currently are no tests available which allow diagnosis of the existence of ischemia in patients prior to tissue necrosis. A pressing requirement for emergency medicine physicians who treat chest pain and stroke symptoms is for a diagnostic test that would enable them to definitively xe2x80x9crule outxe2x80x9d myocardial infarction, stroke, and other emergent forms of ischemia. A need exists for a method for immediate and rapid distinction between ischemic and non-ischemic events, particularly in patients undergoing acute cardiac-type symptoms. The present invention provides such a means.
A broader array of diagnostic tests are available for diagnosis of ischemia in patients with non-acute symptoms. The EKG exercise stress test is commonly used as an initial screen for cardiac ischemia, but is limited by its accuracy rates of only 25-50%. Coronary angiography, an invasive procedure that detects narrowing in the arteries with 90-95% accuracy, is also utilized. Another commonly used diagnostic test is the thallium exercise stress test, which requires injection of radioactive dye and serial tests conducted four hours apart. The present invention, however, has the advantage over the known methods of diagnosis in that it provides equivalent or better accuracy at far lower costs and decreased risk and inconvenience to the patient. The present invention provides specificity and sensitivity levels of 75-95%, which are far more accurate than the EKG exercise stress test and comparable in accuracy to current diagnostic standards. Furthermore, the present invention presents a significant time advantage and is cheaper than competing methods of diagnosis by a factor of at least 15 to 1.
It is known that immediately following an ischemic event, proteins (enzymes) are released into the blood. Well known proteins released after an ischemic heart event include creatine kinase (CK), serum glutamic oxalacetic transaminase (SGOT) and lactic dehydrogenase (LDH). One well known method of evaluating the occurrence of past ischemic heart events is the detection of these proteins in a patient""s blood. U.S. Pat. No. 4,492,753 relates to a similar method of assessing the risk of future ischemic heart events. However, injured heart tissue releases proteins to the bloodstream after both ischemic and non-ischemic events. For instance, patients undergoing non-cardiac surgery may experience perioperative ischemia. Electro-cardiograms of these patients show ST-segment shifts with an ischemic cause which are highly correlated with the incidence of postoperative adverse cardiac events. However, ST-segment shifts also occur in the absence of ischemia; therefore, electrocardiogram testing does not distinguish ischemic from non-ischemic events. The present invention provides a means for distinguishing perioperative ischemia from ischemia caused by, among other things, myocardial infarctions and progressive coronary artery disease.
The present need for rapid, immediate and continuous detection of ischemic states is met by the present invention. Specifically, the present invention provides for a rapid method of testing for the existence of and quantifying ischemia based upon method of detecting and quantifying the existence of an alteration of the serum protein albumin which occurs following an ischemic event. Preferred methods of the present invention for detecting and quantifying this alteration include evaluating and quantifying the cobalt binding capacity of circulating blood, analysis and measurement of the ability of serum albumin to bind exogenous cobalt, detection and measurement of the presence of copper in a purified albumin sample and use of an immunological assay sepcific to the altered form of serum albumin which occurs following an ischemic event. Also taught by the present invention is the use of the compound Asp-Ala-His-Lys-R (SEQ ID NO.1), wherein R is any chemical group capable of being detected when bound to any compound capable of binding to the N-terminus of naturally occurring human albumin (including no additional chemical group), for detection and quantification of an ischemic event.
Advantages and embodiments of the invention include a method for ruling-out the existence of an ischemic state or event in a patient; a method for detecting the existence of asymptomatic ischemia; a method for evaluating patients with angina to rule-out the recent occurrence of an ischemic event; an immediate method for evaluation of patients suffering from chest pain to determine the recent occurrence or non-occurrence of a myocardial infarction.; a method for evaluation of patients suffering from stroke-like signs and symptoms to determine the occurrence or non-occurrence of a stroke and to distinguish between the occurrence of an ischemic stroke and a hemorrhagic stroke; a rapid method for supplementing electrocardiographic results in determining the occurrence of true ischemic events; a method for detecting the occurrence of a true ischemic event in a patient undergoing surgery; a method for evaluating the progression of patients with known ischemic conditions; a method for comparing levels of ischemia in patients at rest and during exercise; a method for assessing the efficacy of an angioplasty procedure; a method for assessing the efficacy of thrombolytic drug therapy; a method for assessing the patency of an in-situ coronary stent; and, a method for detecting in a pregnant woman the occurrence of placental insufficiency.
Additional advantages, applications, embodiments and variants of the invention are included in theDetailed Description of the Invention and Examples sections.
As used herein, the term xe2x80x9cischemic event,xe2x80x9d and xe2x80x9cischemic statexe2x80x9d mean that the patient has experienced a local and/or temporary ischemia due to partial or total obstruction of the blood circulation to an organ. Additionally, the following abbreviations are utilized herein to refer to the following amino acids:
A separate test method for ischemia was described by a common inventor in U.S. Pat. Nos. 5,227,307 and 5,290,519 to Bar-Or et al., herein incorporated by reference in their entirety.
FIG. 1 is a chart illustrating the percentage change in absorbance value in patient samples containing Co ion. The patient samples were taken before percutaneous transluminal coronary angioplasty (PTCA), immediately afger PTCA balloon deflation, 6 hours after the procedure and 24 hours after the procedure, as described in Example 10.
FIG. 2 is a chart setting forth the mean absorbance value in patient samples containing Co ion. The samples were taken before PTCA, immediately after ballon deflation, and 6 and 24 hours after the procedure, as described in Example 10.
FIG. 3 is a chart illustrating the percentage change in absorbance from baseline of patient samples taken before, immediately after and 6 and 24 hours after PTCA for acute myocardial infarction (AMI) and non-AMI patients. This Figure illustrates that AMI patients have elevated ischemia values that do not return to baseline as quickly as those for non-AMI patients following PTCA, as described in Example 10.
FIG. 4 is a chart illustrating the mean change in absorbance from baseline of patient samples taken before, immediately after and 6 and 24 hours after PTCA for AMI and non-AMI patients, as described in Example 10.
FIG. 5 is a chart illustrating the percentage in absorbance of patient samples taken before, immediately after, and 6 and 24 hours after PTCA change in side branch occlusion (SBO) and non-SBO patients, as described in Example 10. This Figure shows that patients with SBO had higher ischemia test values immediately after and 6 hours after PTCA as compared to non-SBO patients.
FIG. 6 is a chart illustrating the percentage change from baseline in absorbance of patient samples taken before coronary stent insertion (with PTCA), immediately after, and 6 and 24 hours after stent insertion, and in patients in which no stent was inserted at the time of PTCA, as described in Example 11.
FIG. 7 is a chrt illustrating the percentage change from baseline in absorbance in patients that experience dysrhythmias during PTCA and in patiens that do not experience dysrhythmias during PTCA, as described in Example 12. FIG. 7 illustrates that patients that experience dysrhythmias during PTCA have higher ischemia test values.
While not being bound by any particular theory, it is believed that the present method works by taking advantage of alterations which occur to the albumin molecule, affecting the N-terminus of albumin during an ischemic (xe2x80x9coxygen-depletedxe2x80x9d) event. (Ischemia occurs when human tissues are deprived of oxygen due to insufficient blood flow.) A combination of two separate phenomena are believed to explain the mechanism by which the ischemia test of the present invention works. First, it is believed that the localized acidosis which occurs during an ischemic event generates free radicals which alter albumin""s N-terminus; thus, by detecting and quantifying the existence of altered albumin, ischemia can be detected and quantified. Second, the acidotic environment present during ischemia results in the release of bound copper (from ceruloplasmin and other copper-containing proteins) which is immediately takenup by albumin. The bound copper also alters the N-terminus of albumin. (Not only does the presence of the complexed copper effectively xe2x80x9calterxe2x80x9d the N-terminus, the metal ion damages the protein structure on binding.) Thus, by detecting and quantifying the existence of altered albumin and/or the copper-albumin complexes, ischemia can be detected and quantified.
The details of the first mechanism are believed to be as follows. In the event of an oxygen insufficiency, cells convert to anaerobic metabolism, which depletes ATP, resulting in localized acidosis and lowered pH, and causing a breakdown in the energy cycle (ATP cycle). Cellular pumps that keep calcium against the gradient are fueled by energy from the ATP cycle. With ATP depletion, the pumps cease to function and cause an influx of calcium into the cell. The excess intracellular calcium activates calcium-dependent proteases (calpain, calmodulin), which in turn cleave segments of xanthine dehydrogenase, transforming the segments into xanthine oxidase. The enzymes involved in this process are membrane-bound and exposed to the outside of the cell, and are thus in contact with circulating blood. Xanthine oxidase generates superoxide free radicals in the presence of hypoxanthine and oxygen. Superoxide dismutase dismutates the oxygen free radicals, turning them into hydrogen peroxide. In the presence of metals such as copper and iron which are found in blood, hydrogen peroxide causes hydroxyl free radicals to be formed. Hydroxyl free radicals in turn cause damage to cells and human tissue. One of the substances damaged by free radicals is the protein albumin, a circulating protein in human blood; specifically believed to be damaged is an amino acid chain within the N-terminus of albumin.
Human serum albumin is the most abundant protein in blood (40 g/l) and the major protein produced by the liver. Many other body fluids also contain albumin. The main biological function of albumin is believed to be regulation of the colloidal osmotic pressure of blood. The amino acid and structure of human albumin have been determined. Specifically, human albumin is a single polypeptide chain consisting of 585 amino acids folded into three homologous domains with one free sulfhydryl group on residue #34. The specific amino acid content of human albumin is:
In one embodiment of the present invention, an excess of cobalt ions are introduced into a purified albumin sample obtained from a patient serum, plasma, fluid or tissue sample. In normal (non-ischemic) patients, cobalt will bind to the amino acid chain on the N-terminus of albumin. In ischemic patients, however, most likely due to the alteration of the binding site of the N-terminus, cobalt binding to albumin is reduced. Accordingly, the occurrence or non-occurrence of an ischemic state can be detected by the presence and quantity of bound or unbound cobalt. Measurement of cobalt can be conducted by atomic absorption, infrared spectroscopy, high-performance liquid chromatography (xe2x80x9cHPLCxe2x80x9d) or other standard or non-standard methods, including radioactive immunoassay techniques.
The details of the second mechanism are believed to be as follows. Ceruloplasmin is a circulating protein which binds copper; approximately ninety-percent of the in vivo copper (copper is abundant in blood, with concentrations comparable to iron) will be bound to ceruloplasmin. The remainder is in other bound forms; almost no free copper exists in circulating blood. In acidic conditions and reduced oxygen conditions, such as happens during ischemia, ceruloplasmin releases some of its bound copper. The released copper is takenup by albumin. Copper and cobalt both bind to albumin at the same site within the N-terminus. Thus, the bound copper, present during ischemia, blocks cobalt from binding to albumin. The decrease in cobalt binding capacity of circulating blood can be measured and quantified as a means for detecting and quantifying the presence of an ischemic event.
A first method of the present invention comprises a method for detecting the occurrence or non-occurrence of an ischemic event in a patient comprising the steps of: (a) contacting a biological sample containing albumin of said patient with an excess quantity of a metal ion salt, said metal ion being capable of binding to the N-terminus of naturally occurring human albumin, to form a mixture containing bound metal ions and unbound metal ions, (b) determining the amount of bound metal ions, and (c) correlating the amount of bound metal ions to a known value to determine the occurrence or non-occurrence of an ischemic event. In this method, said excess quantity of metal ion salt may comprise a predetermined quantity and the quantity of unbound metal ions is detected to determine the amount of bound metal ions. Additionally, the compound selected from the group consisting of Asp-Ala-His-Lys-R (SEQ ID NO.1), wherein R is any chemical group capable of being detected when bound to any compound capable of binding to the N-terminus of naturally occurring human albumin, may be utilized to facilitate detection.
Preferred embodiments of the first method include samples of serum or plasma, or purified albumin. Preferred embodiments also include use of a metal ion salt comprising a salt of a transition metal ion of Groups 1b-7b or 8 of the Periodic Table of the elements, a metal selected from the group consisting of V, As, Co, Sb, Cr, Mo, Mn, Ba, Zn, Ni, Hg, Cd, Fe, Pb, Au and Ag, or cobalt. Also preferred, is detection of the amount of bound metal ions (or, in the case where the excess quantity of metal ion salt is a predetermined quantity, detection of the quantity of unbound metal ions) by atomic absorption or atomic emission spectroscopy or immunological assay. These detection mechanisms are also preferred for determination of the quantity of the compound Asp-Ala-His-Lys-R (SEQ ID NO.1) which is complexed with the metal ion salt in order to detect the quantity of unbound metal ions. A preferred method for conducting said immunological assay is using an antibody specific to an antigen comprising the compound Asp-Ala-His-Lys-R (SEQ ID NO.1), wherein R is said metal ion.
A second method of the present invention is a method of detecting the occurrence or non-occurrence of an ischemic event in a patient comprising the steps of: (a) contacting a biological sample containing albumin of said patient with a predetermined excess quantity of a salt of a metal selected from the group consisting of V, As, Co, Sb, Cr, Mo, Mn, Ba, Zn, Ni, Hg, Cd, Fe, Pb, Au and Ag, to form a mixture containing bound metal ions and unbound metal ions, (b) contacting said mixture with an aqueous color forming compound solution to form a colored solution, wherein said compound is capable of forming color when bound to said metal ion, (c) determining the color intensity of said colored solution to detect the presence of unbound metal ions to provide a measure of bound metal ions, and (d) correlating the amount of bound metal ions to a known value to determine the occurrence or non-occurrence of an ischemic event. Preferred embodiments of this method include the additional step of diluting said colored solution with an aqueous solution isosmotic with blood serum or plasma prior to step (c). Also preferred are: using ferrozine as the color forming compound, and, alternatively, using the compound Asp-Ala-His-Lys-R (SEQ ID NO.1), wherein R is any group capable of forming color when bound to said metal ion as the aqueous color forming compound. Conducting steps (b) and (c) in a pH range of 7 to 9 is preferred. Further, conducting steps (b) and (c) using a spectrophotometer is preferred. Preferred samples in this method also comprise serum, plasma, or purified albumin and a preferred metal ion salt is cobalt.
A third method of the present invention is a method for detecting the occurrence or non-occurrence of an ischemic state in a patient comprising the steps of: (a) detecting the amount of copper ions present in a purified albumin sample of said patient, and (b) correlating the quantity of copper ions present with a known value to determine the occurrence or non-occurrence of an ischemic event. Preferred methods for detection of the amount of copper ions present in the purified albumin sample are by atomic absorption, atomic emission spectroscopy and immunological assay. A preferred method of conducting said immunological assay uses an antibody specific to an antigen comprising the compound Asp-Ala-His-Lys-R (SEQ ID NO.1), wherein R is copper.
A fourth method of the present invention is a method of detecting the occurrence or non-occurrence of an ischemic event in a patient comprising the steps of: (a) contacting a purified albumin sample of said patient, with an aqueous color forming compound solution to form a colored solution, wherein said compound is capable of forming color when bound to copper, (b) determining the color intensity of said colored solution to determine the amount copper in said sample, and (c) correlating the amount of copper to a known value to determine the occurrence or non-occurrence of an ischemic event. Preferred embodiments of this method include the additional step of diluting said colored solution with an aqueous solution isosmotic with blood serum or plasma prior to step (b). Also preferred are: using ferrozine as the color forming compound, and, alternatively, using the compound Asp-Ala-His-Lys-R (SEQ ID NO.1), wherein R is any group capable of forming color when bound to copper ion as the aqueous color forming compound. Conducting steps (a) and (b) in a pH range of 7 to 9 is preferred. Further, conducting step (b) using a spectrophotometer is preferred.
Applications, embodiments and methods of the present invention comprising one or more of the aforementioned four methods of the present invention include: a method for ruling-out the existence of ischemia in a patient, comprising application of either of the aforementioned methods, including application of any of the methods wherein said patient possesses one or more cardiac risk factors, said cardiac risk factors being selected from the group consisting of: age greater than 50, history of smoking, diabetes mellitus, obesity, high blood pressure, high cholesterol, and strong family history of cardiac disease. A variant thereof, comprises subjecting the patient to an exercise treadmill test followed by a second application of the method of claim 1, followed by a comparison of the results of the two applications. This method may be used to detect the existence of ischemia provoked by exercise in an otherwise asymptomatic patient.
Other embodiments, applications and variants of the present invention include a method for ruling-out the occurrence of an temporally-limited ischemic event in a patient comprising application of the method of claim 1; a method of detecting the existence of ischemia in an asymptomatic patient comprising application of the method of claim 1; a method for the evaluation of patients suffering from stroke-like signs to determine the occurrence or non-occurrence of a stroke, comprising application of the method of claim 1; a method for distinguishing between the occurrence of an ischemic stroke and a hemorrhagic stroke, comprising application of the method of claim 1; and a method for assessing the efficacy of an angioplasty procedure, comprising application of the method of claim 1.
The present invention also provides a method for evaluation of a patient presenting with angina or angina-like symptoms to detect the occurrence or non-occurrence of a myocardial infarction, comprising application of the method of claim 1 and application of an electrocardiographic test, followed by correlation of the results of the application of the method of claim 1 with the results of the electrocardiographic test to determine the occurrence or non-occurrence of a myocardial infarction. Preferred electrocardiographic tests are E.C.G., E.K.G. and S.A.E.C.G. tests.
Another method of the present invention is a method for supplementing electrocardiographic results to determine the occurrence or non-occurrence of an ischemic event, comprising application of the method of claim 1 and application of an electrocardiographic test, followed by correlation of the results of application of the method of claim 1 with the results of said electrocardiographic test to determine the occurrence or non-occurrence of an ischemic event. A variant thereof, comprises application of the method wherein said patient is undergoing surgery.
A further method of the present invention is a method for comparing levels of ischemia in patients at rest and during exercise is also taught by the present invention, comprising application of the following steps at designated time intervals: (a) application of the method of claim 1, (b) administration of an exercise treadmill test followed by a second application of the method of claim 1, and (c) comparing the results of the application of the method of claim 1 prior to administration of the exercise treadmill test with the results of the application of the method of claim 1 after administration of the exercise treadmill test, wherein, results obtained from said steps are correlated with results obtained at prior designated time intervals. This embodiment may be used to evaluate patients with known or suspected ischemic conditions, to assess the patency of an in-situ coronary stent and to assess the efficacy of an angioplasty procedure. Preferred designated time intervals are three months, six months or one year.
The present invention also teaches a method for assessing the efficacy of thrombolytic drug therapy, comprising the method of claim 1; a method for detecting in a pregnant woman the occurrence of placental insufficiency, comprising application of the method of claim 1.